Sara Panseri, 1 Alessandra Manzo, 2 Luca Maria Chiesa, 1 and Annamaria Giorgi 2,3. 1. Introduction

Transcription

1 Chemistry Volume 2013, Article ID , 11 pages Research Article Melissopalynological and Volatile Compounds Analysis of Buckwheat Honey from Different Geographical Origins and Their Role in Botanical Determination Sara Panseri, 1 Alessandra Manzo, 2 Luca Maria Chiesa, 1 and Annamaria Giorgi 2,3 1 Department of Veterinary Science and Public Health, UniversitàdegliStudidiMilano,ViaCeloria2,20133Milan,Italy 2 Centre for Applied Studies in the Sustainable Management and Protection of the Mountain Environment, Ge.S.Di.Mont., UniversitàdegliStudidiMilano,ViaMorino8,25048Edolo,Brescia,Italy 3 Department of Agricultural and Environmental Sciences-Production, Landscape, Agroenergy, UniversitàdeglistudidiMilano,ViaCeloria2,20133Milan,Italy Correspondence should be addressed to Sara Panseri; sara.panseri@unimi.it Received 4 June 2013; Revised 5 August 2013; Accepted 20 August 2013 Academic Editor: Jose Alberto Pereira Copyright 2013 Sara Panseri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Volatile organic compounds (VOCs) have been proposed as one of the main factors for differentiating honeys from different botanical/floral origins. In this work, we investigated the volatile profile of honeys, commercially labeled as buckwheat honeys, from the Alps and its relationship with melissopalynological investigation. The results showed that buckwheat honey samples that contained, to different extents, buckwheat pollen grains on melissopalynological analyses showed similar VOCs profiles, distinguishing them from the other honey floral types analyzed. Among VOCs identified, 3-methylbutanal, butanoic acid, pentanoic acid, and isovaleric acid were considerably greater in the buckwheat honey samples from the Alps. Other compounds were identified only in the honeys containing buckwheat pollen grains such as 3-methyl-2-buten-1-ol, 2-butanone, 2-hydroxy-3-pentanone, 4- methylpentanoic acid, 4-pentanoic acid, butanal, 2-methylbutanal, pentanal, dihydro-2-methyl-3(2h)-furanone, 5-methylfurfural, and cis-linalool oxide. These compounds give to buckwheat honey its characteristic aromatic and organoleptic properties and may be considered interesting as potential variety markers for botanical determination. 1. Introduction Honey is a highly energetic sweet food, produced by bees, used by human beings since ancient times, and is of significant economic value today. The organoleptic properties of honey, such as flavour, colour, aroma, and texture are essential factors in consumers estimation of honey quality. These factors are primarily determined by the type of plant species and flowers visited by bees in order to collect nectar or honeydew to produce honey. Climate conditions, bee physiology, honey harvesting and postcollection processing may also influence, to a lesser extent, honey quality. As a consequence, the botanical and, to some extent, also the geographical origins are important characteristics in the evaluation of honey quality [1]. Melissopalynological analyses, consisting of the qualitative and quantitative microscopic examination of honey pollen grains, is, at present, the official test to determine the botanical and geographical origin of honey [2, 3]. Honey containing pollen mainly collected from a single species is classified as monofloral or unifloral, while multifloral honey contains pollen from lots of different species [4]. However, melissopalynology is time consuming, expensive, and highly influenced by the analyst s subjective ability in interpreting data [1, 5]. Moreover, the quantity of pollen found in honey is not always directly correlated with the nectar contribution of a species, since, for example, when the honeys are derived from sterile plants, pollen analysis is of absolutely no use [6]. Considering all these limiting factors, efforts in the identification of alternative or complementary methods to replace or

2 2 Chemistry to integrate pollen analysis are particularly important in order to improve honey quality determination. Studies concerning quality characterization of honey on the basis of phytochemical content in relation with the physicochemical and pollen profile have been published [7 10]. Among phytochemicals, volatile organic compounds (VOCs) have been proposed as one of the main factors for differentiating honeys from different botanical/floral origins [5]. In particular, some unifloral honeys, characterized by individual and specific sensory properties, have been proven to differ one from the other in, among other features, volatile organic composition [11]. Some VOCs are present in the nectar or honeydew collected by bees and could be related to plant characteristics, some others might be originated during honey processing and storage [12, 13]. In this paper, the volatile profile of honeys commercially labeled as buckwheat honeys from the Italian Alps, Russia, Nepal, and Poland was analyzed by means of HS-SPME and GC/MS, a valuable method widely used for volatile extraction and analyses [1, 14, 15]. Buckwheat (Fagopyrum esculentum Moench) is an annual, dicotyledonous plant from the Polygonaceae family with a short growing season [16]. It is a multifood-use pseudocereal. Its inflorescence is formed by 7 9 white, pink, or red blossoms [17]. It is a hermaphroditic species which produces self-incompatible flowers pollinated by insects, including bees. The world s largest producer of buckwheatnowadaysischina,followedbyrussia,ukraine, and France. In Italy, buckwheat cultivation was introduced around the 16th century [18]. However, during the most recent decades cultivation of this crop has strongly declined and today it survives only in a few alpine valleys [19, 20]. Buckwheat honey, collected from the little pink flowers by honeybees during the summer, is characterized by a dark purple color, almost black [21]. Buckwheat monofloral honey is mainly produced in North America (Canada and California),China,andinsomecountriesofEurope,suchasPoland, Russia,Netherlands,andGermany.Becauseofthequite low cultivation of buckwheat plants, in Italy, the monofloral buckwheat honey is difficult to produce and it is usually found as a natural component of multifloral honeys [22]. Buckwheat honey is a high-quality product characterized by a sharp, sweet, and slightly biting taste, having beneficial effects on human health due to its antioxidant, bactericidal, and antiinflammatory properties [23 25]. The effectiveness of VOCs markers in the botanical determination of honeys labeled as buckwheat honeys and their relationship with melissopalynological investigation results are discussed. 2. Materials and Methods 2.1. Honey Samples. Thisstudywascarriedoutoneighttypes of honey samples (Table 1). In 2012, 18 samples were bought from local small-scale beekeepers working in Valtellina, an alpine valley of the Lombardy region. In particular, samples B1 B6 (Table 1), labeled as buckwheat honeys, were produced in Teglio (851 m a.s.l.; min. 352 max m a.s.l.), a mountain village in Valtellina where buckwheat is still cultivated; M1 M6 were reported as multifloral, A1 A3 as acacia honey Table 1: List of samples. Codes, locations, and honey type reported on the commercial label. Sample code Origin Site Honey type on the label B1 B6 Italy Teglio Valtellina-Lombardy region Buckwheat B7 Russia Buckwheat B8-B9 Poland Buckwheat B10 Nepal Buckwheat M1 M3 Italy Teglio Valtellina-Lombardy region Multifloral M4 M6 Italy Valtellina-Lombardy region Multifloral A1 A3 Italy Valtellina-Lombardy region Acacia R1 R3 Italy Valtellina-Lombardy region Rhododendron (producedinanaltitudinalrangeincludedbetween200and 1000 m a.s.l.), and R1 R3 as rhododendron honey (produced over 1000 m a.s.l.). B7, B8, B9, and B10 honey samples, labeled as buckwheat honeys, from Russia, Poland, and Nepal, respectively, were bought from international traders (Gego Enterpriser Pvt. Ltd., Baneshwor, Kathmandu, Nepal; RATOS-NATURA S.C., Olszownica, 75, , Backowice, Swietokrzyskie,Poland;DaryAltaya,OOO,Moscow,Russia). The floral origins of buckwheat samples were verified by using melissopalynological analysis. All samples were stored in darkness at a temperature of 4 6 Cpriortoanalysis Melissopalynological Analysis. Melissopalynological analysis was performed according to the techniques proposed by the International Commission for Bee Botany (ICBB) and published in 1978 [26]. In this study, all honey samples were analyzed to confirm their floral origin. The microscopic analysis of honey sediment composition provides the percentage of the specific pollen observed by microscopic comparison with known pollen grains (Table 1). It is necessary to count at least 300 pollen grains for an estimation of the relative frequencies of pollen types and 500 to 1000 pollen grains for the determination of relative frequencies [27]. The examination under the microscope was carried out at the magnification that was most suitable for identifying the various elements in the sediment (400 to 1000x). After a first general check to ascertain the main types and densities of pollen grains, the relative frequencies of each pollen type were determined. A count of abortive, irregular, or broken pollen grains, fungal spores, hyphae, and microscopic algae, if they could be identified, was performed. If the sediment contained a high percentage of overrepresented pollen, a second count excluding the over-represented pollen was done in order to determine more precisely the relative abundance of the other pollen types. The pollen types present in the honey samples were identified, counted, and classified, according to their percentages, as dominant pollen (more than 45% of the total pollen grains counted), secondary pollen (from 16 to 45%), important minor pollen (from 3 to 15%), and minor pollen (less than 3%) [28].

3 Chemistry HS-SPME Volatile Compounds Sampling from Honey Samples. All the samples were prepared by weighing exactly 5.00 g of honey in a 20 ml glass vial, fitted with cap and equipped with silicon/ptfe septa (Supelco, Bellefonte, PA, USA) and by adding 1 ml of the internal standard solution (IS) in water (1,4-cineol, 1μg/mL, CAS ) to check the qualityofthefibres.attheendofthesampleequilibration period (1 h), a conditioned (1.5 h at 280 C) 50/30 μm Divinylbenzene/Carboxen/polydimethylsiloxane (CAR/PDMS/ DVB) StableFlex fibre (Supelco, Bellefonte, PA) was exposed totheheadspaceofthesamplefortheextraction(180min) by CombiPAL system injector autosampler (CTC analytics, Switzerland).Thefibreandthetimeofextractionusedin this study were selected after preliminary study, and the data were reported in Figure 1.Thebestadsorptionofanalytewas obtained using CAR/PDMS/DVB and 180 min as extraction time. The extraction temperature of 25 Cwasselectedin order to prevent possible matrix alterations (oxidation of some compounds, particularly aldehydes and furans). To keep a constant temperature during analysis, the vials were maintained on a heater plate (CTC Analytics, Zwingen, Switzerland). As demonstrated in other researches in which the VOCs profile of food is investigated, the use of high extraction temperature can lead to ex novo formation of volatile compounds or to the production of artefacts [29, 30] Gas Chromatography-Mass Spectrometry Analysis of VOCs. HS-SPME analysis was performed using a Trace GC Ultra (Thermo-Fisher Scientific, Waltham, MA, USA) Gas Chromatograph coupled to a quadrupole Mass Spectrometer Trace DSQ (Thermo-Fisher Scientific, Waltham, MA, USA) and equipped with an Rtx-Wax column (30 m; 0.25 mm i.d.; 0.25 μm film thickness, Restek, USA). The oven temperature program was: from 35 C, hold 8 min, to 60 Cat4 C/min, then from 60 Cto160 Cat6 C/min, and finally from 160 Cto 200 Cat20 C/min. Carryover and peaks originating from the fibre were regularly assessed by running blank samples. After each analysis, fibres were immediately thermally desorbed in the GC injector for 5 min at 250 C to prevent contamination. The injections were performed in splitless mode (5 min). The carrier gas was helium at a constant flow of 1 ml 1.Thetransfer line to the mass spectrometer was maintained at 230 C, andtheionsourcetemperaturewassetat250 C. The mass spectra were obtained by using a mass selective detector with the electronic impact at 70 ev, a multiplier voltage of 1456 V, and by collecting the data at rate of 1 scan s 1 over the m/z rangeof Compoundswereidentifiedby comparing the retention times of the chromatographic peaks with those of authentic compounds analyzed under the same conditions when available. The identification of MS fragmentation patterns was performed either by comparison with those of pure compounds or using the National Institute of Standards and Technology (NIST) MS spectral database. Volatile compounds measurements from each headspace of honey extracts were carried out by peak area normalization (expressed in percentage). All analyses were done in duplicate. Total peak areas (a.u.) CAR/PDMS/DVB CAR/PDMS PDMS/DVB Extraction time (min) PDMS PA Figure 1: Total absorption peak areas (arbitrary unit) for CAR/ PDMS/DVB, CAR/PDMS, PDMS/DVB, PDMS, and PA fibres at different extraction time (15, 45, 60, 90, 120, 180, and 240 min). 3. Results and Discussion 3.1. Melissopalynological Analysis. The results of the melissopalynological analyses of the honeys labeled as buckwheat honeys are reported in Tables 2 and 3. Theresultsshowed that the buckwheat honey samples B1, B2, and B3 could be classified as monofloral with 45.5%, 52%, and 46% of buckwheat pollen. In Valtellina, the maximum altitude at which buckwheatiscultivatedis1200ma.s.l.,anditsflowering period is August, thus, giving an indication on the elevation and the period of honey samples B1, B2, and B3 production. On the contrary, honey samples B4, B5, and B6, also labeled as buckwheat honeys, had to be classified as multifloral, with a very low (5%, 4.5%, and 5.6%, resp.) relative frequency of buckwheat pollen. The high presence of pollen grains of plants belonging to the Rhododendron genus, growing at an elevation between 1600 and 2300 m a.s.l. and flowering from June to mid-july, suggested that honey samples B4 and B5 were produced in Valtellina at higher altitude and in a different season compared to honey samples B1, B2, and B3. The high presence of pollen grains of Eryngium alpinum L. and Euphrasia officinalis L. in honey sample B6 also suggested that it was produced at higher altitude compared to honey samples B1, B2, and B3. Finally,thepresenceofpollengrainsofplantsbelonging to the Clematis genus, flowering from May to July, and Lotus alpinus (DC.) Schleicher, growing from 1700 to 2700 m a.s.l. and flowering in July, seemed to confirm the different period and environment of production of honey samples B4, B5, and B6. The Polish (B8 and B9) and Nepali (B10) samples had to be classified as multifloral honeys with a prevalence of buckwheat pollen grain (25%, 30%, and 16%, resp.), while the Russian sample (B7) had to be classified as multifloral with 5% of buckwheat honey. The presence of pollen grains from

4 4 Chemistry Table 2: Relative frequencies of the main pollen types in honeys labeled as buckwheat honeys. B1, B2, and B3: buckwheat honey from Italy (Valtellina); B4, B5, and B6: buckwheat honey from Italy (Teglio). Dominant pollen (>45%) Secondary pollen (16 45%) Important minor pollen (3 15%) Minor pollen (<3%) Sample code B1 B2 B3 B4 B5 B6 F. esculentum 45.5% F. esculentum 52% F. esculentum 46% Robinia 16.5% Trifolium repens 10.3% Robinia 8.5% Tilia 3% Sedum/Sempervivum 2% Prunus 2.8% Gleditsia 2.2% Pyrus/Malus 1.9% Acer 1.5% Salix 0.7% Clematis 0.4% Trifolium pratense <1% Umbelliferae <1% Trifolium repens 8.7% Hedera 5.2% Verbena 2% Castanea 1.8% Pyrus/Malus 1% Salix 0.4% Sedum/Sempervivum <1% Knautia/Scabiosa <1% Salix 8% Rubus 4.7% Tilia 2% Ericaceae 2% Compositae 1.8% Trifolium repens 1.5% Umbelliferae <1% Achillea spp. <1% Melilotus 79% Rhododendron 36% Clematis 7.2% Clematis 8.3% F. esculentum 5% Lotus alpinus 5.4% Trifolium repens 3% Prunus 2.6% Ranunculaceae 1.7% Robinia 1% Campanulaceae 0.9% Trifolium pretense 0.7% Acer 0.7% Ranunculaceae 0.7% Umbelliferae 0.4% Salvia <1% Sedum/Sempervivum <1% Rhododendron 21% F. esculentum 4.5% Rubus 4% Mentha 4% Eryngium alpinum 4% Compositae 2% Lotus alpinus 2% Prunus 1.8% Compositae 1% Trifolium repens <1% Salix <1% Euphrasia officinalis 37% Compositae 18% Eryngium alpinum 17.8% F. esculentum 5.6% Mentha 8% Clematis 5% Rubus 4.3% Prunus 2% Umbelliferae 1.8% Hedera 0.9% Salvia 0.2% Table 3: Relative frequencies of the main pollen types in honeys labeled as buckwheat honeys. B7: buckwheat honey from Russia; B8 and B9: buckwheat honey from Poland; B10: buckwheat honey from Nepal. Dominant pollen (>45%) Secondary pollen (16 45%) Important minor pollen (3 15%) Minor pollen (<3%) Sample code B7 B8 B9 B10 Cruciferae 67% Helianthus 30% Melilotus 24% Cruciferae 9% Fagopyrum esculentum 5% Trifolium repens 5% Echium 4% Verbascum 4% Robinia 3% Labiatae 2% Onobrychis 2% Cynoglossum 1% Prunus 1% Campanulaceae 1% Compositae 1% Lotus 1% Umbelliferae 1% Fagopyrum esculentum 25% Brassica napus 14% Echium 8% Trifolium repens 4% Helianthus 3% Umbelliferae 3% Lotus 2% Trifolium repens 1% Compositae <1% Sedum/Sempervivum <1% Fagopyrum esculentum 30% Brassica napus 17.9% Echium 13% Gleditsia 12.4% Helianthus 4.8% Sedum/Sempervivum 3% Lamium 2.3% Trifolium repens 1.9% Salix 1.5% Lotus corniculatus 1.5% Umbelliferae 1.5% Salvia 1.5% Euphorbiaceae <1% Fagopyrum esculentum 16% Helianthus 4% Compositae 4% Eucalyptus 3% Rosaceae 2% Umbelliferae 2% Bombacaceae 1% Castanea 1% Labiatae <1%

5 Chemistry 5 plants of the Bombacaceae genus in the Nepali honey sample confirmed its Asiatic origin, suggesting it was produced in atropicalorsubtropicalareaofnepal,whereplantsofthis genus grow. Finally, as Poland is one of the major producers of canola in Europe, the presence of a relevant quantity of Brassica napus L. pollen grains in honey samples B9 and B10 was coherent with its declared origin. The results regarding the other honey samples (M1 M6, A1 A3, and R1 R3) confirmed the botanical classification reported on the commercial label (data not shown), no buckwheat pollen grain were recovered Analysis of VOCs in Honey Samples. Honey volatiles are a very complex mixture of substances frequently occurring at a very low concentration and with poor chemical stability. Thus, as reported by many authors [2, 31 33], the use of headspace solid-phase-microextraction (HS-SPME) and gaschromatography/mass-spectrometry (GC/MS), a very sensible and solvent-free method for extraction and analyses of this chemical fraction, is particularly suitable. In our experimental conditions, 86 compounds have overall been identified in the honey samples analyzed, and they are summarized in Tables 4 and 5. These compounds belonged to different major chemical classes as follows: alcohols, phenols, ketones, free fatty acid, esters, aldehydes, furans, and terpenes. This paper is the first investigation on the VOCs profile of a buckwheat monofloral honey from Valtellina (North of Italy). Remarkable differences in VOCs profiles were observed when comparing honey samples of different floral origins. Consistent with other authors [34 36], most of the compoundswereidentifiedinalloftheanalyzedhoneys, but the proportion in which they occurred appeared very different taking into account the different floral/botanical origin. Similarly, in each of the analyzed samples there were compounds which were not present in other types of honeys to be evaluated as potential floral markers. The VOCs profile of honeys labeled as buckwheat honeys (B1 B10) was similar, particularly for samples containing a relevant quantity of buckwheat pollen grains despite the different geographical origin. In addition, comparing it with thevocsprofileoftheotherhoneysamples,notcontaining buckwheat pollen grains (M1 M6, A1 A3, and R1 R3), some differences were identified, particularly regarding the composition and the concentration of some chemical classes such as alcohols, aldehydes, free fatty acids, furans, and terpenes as shown in Tables 4 and 5. Buckwheat honey is characterized by a sharp, sweet, and slightly biting taste, and its organoleptic characteristics have been proven to be reflected in its composition and concentrations of volatile compounds [33]. Moreover, among the aldehydes, methylbutanals have been reported to be responsible for the characteristic pungent, sweetish, and malty flavour of buckwheat honey [22, 37]. In our experimental condition, 3-methylbutanal was found in highest concentration in buckwheat honey, and 2- methylbutanal was found to be present only in the honeys containing buckwheat pollen grains. These compounds are commonly found in barley malt [38]. They are known to be Strecker aldehydes, and their presence in honeys is usually associated with the Maillard browning reactions. The extremely high amounts of methylbutanals in buckwheat honeys compared with some other honeys suggested that this type of honey contains a higher abundance of Strecker degradation precursors, such as amino acids, which would result in a honey with an aroma resembling that which develops upon heat-promoted chemical reactions that occur during the malting of barley [22]. The presence of other Maillard reaction products such as phenylacetaldehyde and dimethyl sulfide supports this hypothesis. As reported in the literature [33], besides aldehydes, also the concentrations of free fatty acids, like butanoic acid and pentanoic acid, were considerably greater in the honey samples B1, B2, B3, and B9, those containing the higher quantity of buckwheat pollen grains. Butanoic acid gives buckwheat honey its typical pungent smell and pentanoic acid has a rancid smell and an acid taste [37]. Such chemical compounds, characterized by high concentrations in a honey type and specific sensory properties, are to be considered interesting as potential variety markers [33]. Wolski et al. [32]reportedbutanal,phenol, trans-linalool oxide, 3,4,5-trimethylphenol as buckwheat honey marker compounds because they were not found in other honey types. In this study, only butanal was confirmed to be such a marker, being present only in the two honey samples containing a relevant quantity of buckwheat pollen grains, corresponding to the Italian monofloral buckwheat honey (samples B1, B2, and B3) and the Polish honeys (samples B8 and B9). Inthesamehoneysamples,wealsofoundsignificantly greatquantitiesofisovalericacid,neverreportedbeforein buckwheathoney.isovalericacidisapotent,odorant,volatile compound, exhibiting an unpleasant odor associated with the rank smell of perspiring feet and has been considered to be an important off-flavor compound in honeys [39]. Isovaleric acid, has been found to be an important odorant for Anarcardium occidentale L. andcroton sp. honeys from Brazil [40]. Finally, we have identified additional characteristic volatile compounds of buckwheat honey such as 3-methyl-2- buten-1-ol, 2-butanone, 2-hydroxy-3-pentanone, 4-methylpentanoic acid, 4-pentanoic acid, butanal, 2-methylbutanal, pentanal, dihydro-2-methyl-3(2h)-furanone, 5-methylfurfural, and cis-linalool oxide. Pasini et al. [21] reported 5- methylfurfural and other furans as important buckwheat honey marker compounds. In the literature, furanic compounds were reported to derive from sugar degradation and considered to be indicators of thermal processes and storage [41]. 4. Conclusion Honey samples labeled as buckwheat honey, found to contain, to different extents, buckwheat pollen grains on melissopalynological analyses, show similar VOCs profiles, distinguishing them from the other honey floral types analyzed. In particular, the honey samples from the Italian Alps, classified as monofloral buckwheat honey, and the two samples from

6 6 Chemistry Table 4: Volatile compounds identified in investigated honey samples expressed as percentages. B1 B6: buckwheat honey from Italy (Teglio- Valtellina); B7: buckwheat honey from Russia; B8 and B9: buckwheat honey from Poland; B10: Buckwheat honey from Russia. Compounds RT a Identification b Sample code B1 c B2 c B3 c B4 c B5 c B6 c B7 d B8 d B9 d B10 d Alcohols Ethanol 3.97 MS, LRI ,3-Butanedione 4.98 MS, LRI Methyl-3-buten-2-ol 7.39 MS, LRI nd 0.05 nd 1-Butanol MS, LRI Methylbutanol MS, LRI Methylpropanol MS, LRI nd nd nd nd nd 3-Methylbutanol MS, LRI Methyl-2-buten-1-ol MS, LRI nd Methyl-2-buten-1-ol MS, LRI Methylbutenol MS, LRI nd nd 0.03 nd Total Phenols 2-Caren-10-al MS, LRI nd nd 0.14 p-cymen-8-ol MS, LRI Phenol MS, LRI nd p-cresol MS, LRI nd Thymol MS, LRI nd nd nd nd nd nd nd nd Total Ketones 2-Butanone 6.61 MS, LRI nd nd nd nd nd Hydroxy-2-butanone MS, LRI Hydroxyacetone MS, LRI Hydroxy-3-pentanone MS, LRI Hydroxy-2-butanone MS, LRI Methyl-butyrolactone MS, LRI nd 4-Oxoisophorone MS, LRI Total Free fatty acid Acetic acid MS, LRI Formic acid MS, LRI nd nd Propanoic acid MS, LRI Isobutyric acid MS, LRI Butanoic acid MS, LRI Propanoic acid MS, LRI nd nd nd nd nd nd nd nd nd nd Isovaleric acid MS, LRI Pentanoic acid MS, LRI Butenoic acid MS, LRI Methylpentanoic acid MS, LRI Methylpentanoic acid MS, LRI Pentanoic acid MS, LRI Benzyl nitrile MS, LRI nd 0.01 nd Heptanoic acid MS, LRI Octanoic acid MS, LRI Nonanoic acid MS, LRI nd nd nd nd Benzoic acid MS, LRI Total Esters Ethyl acetate 3.04 MS, LRI nd Butanoic acid 3-methyl-ethyl ester 8.60 MS, LRI nd nd 0.01 nd nd nd nd nd nd nd Ethyl lactate MS, LRI nd 0.03 nd Vinyl 2,2-dimethyl pentanoate MS, LRI nd nd nd nd nd nd nd nd nd 0.13 Propylene carbonate MS, LRI 0.01 nd 0.01 nd nd nd nd nd nd nd Total

7 Chemistry 7 Table 4: Continued. Compounds RT a Identification b Sample code B1 c B2 c B3 c B4 c B5 c B6 c B7 d B8 d B9 d B10 d Aldehydes Acetaldehyde 1.75 MS, LRI Butanal 2.86 MS, LRI Methylbutanal 3.37 MS, LRI Methylbutanal 3.45 MS, LRI nd Pentanal 4.79 MS, LRI Hexanal 9.01 MS, LRI nd nd nd nd nd nd nd nd nd Pentanal MS, LRI nd nd nd nd nd nd nd Heptanal MS, LRI nd nd nd nd nd nd 0.07 nd nd 0.05 Nonanal MS, LRI nd Benzaldehyde MS, LRI Lilac aldehyde A MS, LRI nd nd nd nd nd nd nd nd nd nd Lilac aldehyde B MS, LRI nd nd nd nd nd Benzeneacetaldehyde MS, LRI nd Nicotinaldehyde MS, LRI nd nd nd nd nd Methyl-2-octenedial MS, LRI nd nd nd nd nd 0.15 Phenylacetaldehyde MS, LRI nd nd nd nd nd nd 0.05 nd nd Pyrrolecarboxaldehyde MS, LRI nd Cinnamaldehyde MS, LRI nd nd nd nd nd nd nd nd nd nd Total Furans Furan 2.19 MS, LRI nd nd nd nd nd nd nd nd nd 0.68 Methylfuran 3.15 MS, LRI nd nd nd nd nd nd 0.28 nd nd 1.19 Dihydro-2-methyl-3(2H)-furanone MS, LRI Furfural MS, LRI (2-Furanyl)-ethanone MS, LRI nd 5-Methylfurfural MS, LRI Dihydro-5-methyl-2(3H)-furanone MS, LRI nd nd nd nd nd nd nd Dihydro-3-methyl-2(3H)-furanone MS, LRI nd nd nd nd nd (5H)-Furanone MS, LRI ,5-Dimethyl-2-furaldehyde MS, LRI nd Total Terpenes Verbenene MS, LRI nd nd nd nd nd nd nd nd nd 0.03 α-phellandrene MS, LRI nd nd nd nd nd nd nd nd nd 0.03 α-terpinene MS, LRI nd nd nd nd nd nd nd τ-terpinene MS, LRI nd nd nd nd nd 0.07 Cymene MS, LRI nd nd nd nd nd nd 0.65 nd nd 0.08 cis-linalool oxide MS, LRI trans-linalool oxide MS, LRI nd nd Menthofuran MS, LRI nd nd nd nd nd nd nd nd nd 0.25 Linalool MS, LRI Damascenone MS, LRI nd nd nd nd nd nd α-terpinolene MS, LRI nd nd nd nd nd nd 0.24 Carvacrol MS, LRI nd 0.06 nd nd nd nd nd nd nd 0.09 Total Miscellaneous Dimethyl sulfide 1.93 MS, LRI Eugenol MS, LRI 0.01 nd nd nd nd nd nd nd nd nd Total a Retention time; b MS: mass spectrum tentatively identified using NIST 05 and Wiley 275 libraries; LRI: linear retention index. c Normalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of buckwheat honeys from Italy (Valtellina). d Normalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of buckwheat honeys from Russia, Poland, and Nepal. nd: not detected.

8 8 Chemistry Table 5: Volatile compounds identified in investigated honey samples expressed as percentages. M1 M3: multifloral honeys from Italy (Teglio- Valtellina); M4 M6: multifloral honeys from Italy (Valtellina); A1 A3: acacia honeys from Italy (Valtellina); R1 R3: rhododendron honeys from Italy (Valtellina). Compounds RT a Identification b Sample code M1 c M2 c M3 c M4 c M5 c M6 c A1 c A2 c A3 c R1 c R2 c R3 c Alcohols Ethanol 3.97 MS, LRI ,3-Butanedione 4.98 MS, LRI 2.97 nd 0.21 nd Methyl-3-buten-2-ol 7.39 MS, LRI nd nd nd Butanol MS, LRI Methylbutanol MS, LRI Methylpropanol MS, LRI nd nd 0.05 nd nd nd nd nd nd 3-Methylbutanol MS, LRI Methyl-2-buten-1-ol MS, LRI Methyl-2-buten-1-ol MS, LRI nd nd nd nd nd nd nd nd nd nd nd nd 2-Methylbutenol MS, LRI nd nd 0.01 Total Phenols 2-Caren-10-al MS, LRI 0.09 nd nd nd nd nd nd nd nd nd p-cymen-8-ol MS, LRI nd nd nd Phenol MS, LRI nd p-cresol MS, LRI nd nd nd nd 0.01 Thymol MS, LRI nd 0.01 nd nd nd Total Ketones 2-Butanone 6.61 MS, LRI nd nd nd nd nd nd nd nd nd nd nd nd 3-Hydroxy-2-butanone MS, LRI Hydroxyacetone MS, LRI nd Hydroxy-3-pentanone MS, LRI nd nd nd nd nd nd nd nd nd nd nd nd 1-Hydroxy-2-butanone MS, LRI nd Methyl-butyrolactone MS, LRI nd 0.05 nd Oxoisophorone MS, LRI nd nd Total Free fatty acid Acetic acid MS, LRI Formic acid MS, LRI Propanoic acid MS, LRI Isobutyric acid MS, LRI Butanoic acid MS, LRI Propanoic acid MS, LRI nd 0.01 nd nd Isovaleric acid MS, LRI Pentanoic acid MS, LRI nd 0.02 nd 2-Butenoic acid MS, LRI nd 0.01 nd nd nd nd Methylpentanoic acid MS, LRI Methylpentanoic acid MS, LRI nd nd nd nd nd nd nd nd nd nd nd nd 4-Pentanoic acid MS, LRI nd nd nd nd nd nd nd nd nd nd nd nd Benzyl nitrile MS, LRI nd 0.02 nd nd nd 0.01 nd nd nd nd Heptanoic acid MS, LRI Octanoic acid MS, LRI nd nd Nonanoic acid MS, LRI nd 0.02 nd 0.02 nd nd Benzoic acid MS, LRI Total Esters Ethyl acetate 3.04 MS, LRI nd nd nd nd nd nd nd nd nd Butanoic acid 3-methyl-ethyl ester 8.60 MS, LRI nd nd 0.03 nd nd 0.03 nd nd nd Ethyl lactate MS, LRI nd 0.01 nd 0.01 nd 0.02 nd 0.02 nd 0.04 Vinyl 2,2-dimethyl pentanoate MS, LRI nd 0.1 nd nd nd nd nd nd nd Propylene carbonate MS, LRI nd nd nd nd nd nd nd nd nd Total nd

9 Chemistry 9 Table 5: Continued. Compounds RT a Identification b Sample code M1 c M2 c M3 c M4 c M5 c M6 c A1 c A2 c A3 c R1 c R2 c R3 c Aldehydes Acetaldehyde 1.75 MS, LRI nd nd nd Butanal 2.86 MS,LRI nd nd nd nd nd nd nd nd nd nd nd nd 2-Methylbutanal 3.37 MS,LRI nd nd nd nd nd nd nd nd nd nd nd nd 3-Methylbutanal 3.45 MS, LRI nd nd nd nd nd nd nd nd nd Pentanal 4.79 MS,LRI nd nd nd nd nd nd nd nd nd nd nd nd Hexanal 9.01 MS, LRI nd nd nd nd nd nd 0.04 nd 0.07 nd nd Pentanal MS, LRI nd nd nd nd nd nd nd nd nd nd Heptanal MS, LRI nd nd nd nd nd nd nd Nonanal MS, LRI nd nd nd nd nd nd Benzaldehyde MS, LRI Lilac aldehyde A MS, LRI nd nd nd Lilac aldehyde B MS, LRI nd Benzeneacetaldehyde MS, LRI nd 0.01 nd Nicotinaldehyde MS, LRI nd 0.04 nd nd nd nd Methyl-2-octenedial MS, LRI nd nd nd Phenylacetaldehyde MS, LRI 0.03 nd 0.07 nd nd nd nd 0.02 nd nd nd nd 2-Pyrrolecarbaldehyde MS, LRI nd 0.03 nd 0.01 nd nd Cinnamaldehyde MS, LRI 0.03 nd nd 0.02 nd 0.02 nd Total Furans Furan 2.19 MS, LRI 0.18 nd 0.03 nd 0.02 nd nd nd nd nd nd nd Methylfuran 3.15 MS, LRI nd nd nd nd Dihydro-2-methyl-3(2H)-furanone MS, LRI nd nd nd nd nd nd nd nd nd nd nd nd Furfural MS, LRI (2-Furanyl)-ethanone MS, LRI nd Methylfurfural MS,LRI nd nd nd nd nd nd nd nd nd nd nd nd Dihydro-5-methyl-2(3H)-furanone MS, LRI 0.75 nd nd nd 0.01 Dihydro-3-methyl-2(3H)-furanone MS, LRI nd nd nd (5H)-Furanone MS, LRI nd 0.03 nd nd nd nd nd 4,5-Dimethyl-2-furaldehyde MS, LRI nd 0.02 nd Total Terpenes Verbenene MS, LRI nd nd nd nd nd nd nd nd nd nd α-phellandrene MS, LRI 0.03 nd 0.05 nd 0.03 nd nd nd nd nd nd nd α-terpinene MS, LRI nd nd nd nd nd τ-terpinene MS, LRI nd nd nd nd nd nd nd 0.01 nd nd nd nd Cymene MS, LRI nd 0.01 nd nd nd nd nd nd nd cis-linalool oxide MS, LRI nd nd nd nd nd nd nd nd nd nd nd nd trans-linalool oxide MS, LRI nd Menthofuran MS, LRI 0.26 nd 0.50 nd 0.10 nd nd nd nd nd nd nd Linalool MS, LRI nd Damascenone MS, LRI nd 0.01 nd 0.01 nd 0.02 nd α-terpinolene MS, LRI 0.14 nd 0.25 nd nd nd nd Carvacrol MS, LRI nd nd 0.01 nd nd nd nd nd nd nd Total Miscellanous Dimethyl sulfide 1.93 MS, LRI nd 1.00 nd nd nd nd Eugenol MS, LRI nd 0.01 nd nd nd nd nd Total nd a Retention time; b MS: mass spectrum tentatively identified using NIST 05 and Wiley 275 libraries; LRI: linear retention index. c Normalized amount of volatile compounds (percentage) (peak of volatile compound/total peak area of all volatile compounds) of multifloral, acacia, and rhododendron honey from Italy (Valtellina). nd: not detected.

10 10 Chemistry Poland, classified as multifloral honey but containing a significant level of buckwheat pollen grains, were found to have a very similar volatile organic compounds profile, despite the different geographical origin. Thus, the VOCs profile analyses seemed to be useful in distinguishing honeys containing buckwheat pollen grains from those of different botanical origin. Many volatile compounds were identified in all honey types, but in the honeys containing buckwheat pollen grains there were components that were not present in other honey types such as 3-methyl-2-buten-1-ol, 2-butanone, 2-hydroxy- 3-pentanone, 4-methylpentanoic acid, 4-pentanoic acid, butanal, 2-methylbutanal, pentanal, dihydro-2-methyl-3(2h)- furanone, 5-methylfurfural, and cis-linalool oxide. Among them, butanal and 2-methylbutanal have been proposed as buckwheat honey markers also by other authors [22, 32, 33]. According to the literature, butanoic acid and pentanoic acid were considerably greater in the buckwheat honey samples particularly in those from Italy and Poland containing the higher level of buckwheat pollen grains. These compoundshavebeenreportedtogivebuckwheathoneyits characteristic aromatic and organoleptic properties and are to be considered interesting as potential variety markers. Finally, isovaleric acid, whose presence is reported to have a negative sensorial impact, was for the first time detected in buckwheat honey, particularly in the Italian monofloral buckwheat honeys and in the Polish samples. Conflict of Interests The authors declare that they have no conflict of interests. Acknowledgments ThisstudywaspartlysupportedbyagrantfromthePICInterreg Italia-Svizzera Proalpi-valori e sapori delle produzioni tradizionali alpine and partly by Accordo di Programma, affermazione in Edolo del Centro di Eccellenza Università della Montagna MIUR-Università degli Studi di Milano, protocol no /08/2011. References [1] M. V. Baroni, M. L. Nores, M. D. P. Díaz et al., Determination of volatile organic compound patterns characteristic of five unifloral honey by solid-phase microextraction-gas chromatographymass spectrometry coupled to chemometrics, Agricultural and Food Chemistry,vol.54,no.19,pp ,2006. [2] E. Alissandrakis, D. Daferera, P. A. Tarantilis, M. Polissiou, and P. C. Harizanis, Ultrasound-assisted extraction of volatile compounds from citrus flowers and citrus honey, Food Chemistry, vol. 82, no. 4, pp , [3] G. Aronne and V. de Micco, Traditional melissopalynology integrated by multivariate analysis and sampling methods to improve botanical and geographical characterisation of honeys, Plant Biosystems,vol.144,no.4,pp ,2010. [4] S. Ampuero, S. Bogdanov, and J.-O. Bosset, Classification of unifloral honeys with an MS-based electronic nose using different sampling modes: SHS, SPME and INDEX, European Food Research and Technology,vol.218,no.2,pp ,2004. [5] I. Stanimirova, B. Üstün, T. Cajka et al., Tracing the geographical origin of honeys based on volatile compounds profiles assessment using pattern recognition techniques, Food Chemistry,vol.118,no.1,pp ,2010. [6] C.Guyot,V.Scheirman,andS.Collin, Floraloriginmarkers of heather honeys: Calluna vulgaris and Erica arborea, Food Chemistry,vol.64,no.1,pp.3 11,1999. [7]A.C.Soria,M.González, C. de Lorenzo, I. Martínez-Castro, and J. Sanz, Characterization of artisanal honeys from Madrid (Central Spain) on the basis of their melissopalynological, physicochemical and volatile composition data, Food Chemistry,vol.85,no.1,pp ,2004. [8] V. Kaškoniene and P. R. Venskutonis, Floral markers in honey of various botanical and geographic origins: a review, Comprehensive Reviews in Food Science and Food Safety,vol.9, no. 6, pp , [9] A. Giorgi, M. Madeo, J. Baumgartner, and G. C. Lozzia, The relationships between phenolic content, pollen diversity, physicochemical information and radical scavenging activity in honey, Molecules,vol.16,no.1,pp , [10] F. Tornuk, S. Karaman, I. Ozturk et al., Quality characterization of artisanal and retail Turkish blossom honeys: determination of physicochemical, microbiological, bioactive properties and aroma profile, Industrial Crops and Products, vol. 46, pp , [11] I. Jerković and Z. Marijanović, Volatile composition screening of Salix spp. nectar honey: benzenecarboxylic acids, norisoprenoids, terpenes, and others, Chemistry and Biodiversity, vol. 7,no.9,pp ,2010. [12] F. Bianchi, M. Careri, and M. Musci, Volatile norisoprenoids as markers of botanical origin of Sardinian strawberry-tree (Arbutus unedo L.) honey: characterisation of aroma compounds by dynamic headspace extraction and gas chromatography-mass spectrometry, Food Chemistry,vol.89, no.4,pp , [13] J. S. Bonvehí and F. Ventura, Flavour index and aroma profiles of fresh and processed honeys, the Science of Food and Agriculture, vol. 83, no. 4, pp , [14] I. Jerković, Z. Marijanović, J. Kezić, and M. Gugić, Headspace, volatile and semi-volatile organic compounds diversity and radical scavenging activity of ultrasonic solvent extracts from Amorpha fruticosa honey samples, Molecules,vol.14,no.8,pp , [15] W. Wardencki, T. Chmiel, T. Dymerski, P. Biernacka, and B. Plutowska, Application of gas chromatography, mass spectrometry and olfactometry for quality assessment of selected food products, Ecological Chemistry and Engineering S, vol.16,pp , [16] T. Björkman, Guide To Buckwheat Production in the Northeast. N.Y. State Agr. and Ext. Services, Cornell University, Geneva, NY, USA, [17] D. Janovská, J. Kalinová, and A. Michalová, Metodika pestování pohanky v ekologickém a konvencním zemedelství. Metodika pro praxi, Výzkumný ústav rostlinné výroby, Prague, Czech Republic, [18] I. Fontanari, Un antico cereale da riscoprire: il grano saraceno, Terra Trentina,vol.5,pp.46 47,2004. [19] B. Borghi, C. Minoia, M. Cattaneo et al., Primi risultati di prove agronomiche e varietali, L Informatore Agrario,vol.25,pp.59 63, [20] B. Borghi, Il grano saraceno, coltura che ha futuro, Vita in Campagna, vol. 10, pp , 1996.

11 Chemistry 11 [21] F. Pasini, S. Gardini, G. L. Marcazzan, and M. F. Caboni, Buckwheat honey: screening of composition and properties, Food Chemistry, vol. 141, pp , [22] R. Dalby, Classic American honey plant: buckwheat, American Bee Journal,vol.140,pp ,2000. [23] J.Zhou,P.Li,N.Chengetal., Protectiveeffectsofbuckwheat honey on DNA damage induced by hydroxyl radicals, Food and Chemical Toxicology,vol.50,no.8,pp ,2012. [24] K. Brudzynski, K. Abubaker, and T. Wang, Powerful bacterial killing by buckwheat honeys is concentration-dependent, involves complete DNA degradation and requires hydrogen peroxide, Frontiers in Microbiology, vol. 3, p. 242, [25] C. E. Manyi-Loh, A. M. Clarke, and R. N. Ndip, An overview of honey: therapeutic properties and contribution in nutrition and human health, African Microbiology Research, vol. 5, no. 8, pp , [26] J. Louveaux, A. Maurizio, and G. Vorwhol, Method of Mellissopalynology, International Commission for Bee Botany of IUBS, [27] G. Ricciardelli D albore and L. Persano Oddo, Flora Apistica Italiana, Istituto sperimentale per la zoologia agraria, [28] O. M. Barth, Microscopical analysis of honey samples, Anais da Academia Brasileira de Ciências, vol.42,no.2,pp , 1970 (Portuguese). [29] S. Panseri, S. Soncin, L. M. Chiesa, and P. A. Biondi, A headspace solid-phase microextraction gas-chromatographic massspectrometric method (HS-SPME-GC/MS) to quantify hexanal in butter during storage as marker of lipid oxidation, Food Chemistry,vol.127,no.2,pp ,2011. [30] S. Soncin, L. M. Chiesa, S. Panseri, P. Biondi, and C. Cantoni, Determination of volatile compounds of precooked prawn (Penaeus vannamei)and cultured gilthead sea bream (Sparus aurata) stored in ice as possible spoilage markers using solid phase microextraction and gas chromatography/mass spectrometry, the Science of Food and Agriculture,vol.89, no. 3, pp , [31] G. Bentivenga, M. D Auria, P. Fedeli, G. Mauriello, and R. Racioppi, SPME-GC-MS analysis of volatile organic compounds in honey from Basilicata. Evidence for the presence of pollutants from anthropogenic activities, International Journal of Food Science and Technology, vol.39,no.10,pp , [32] T. Wolski, K. Tambor, H. Rybak-Chmielewska, and B. Kędzia, Identification of honey volatile components by solid phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS), Apicultural Science, vol.50,no. 2, pp , [33] B. Plutowska, T. Chmiel, T. Dymerski, and W. Wardencki, A headspace solid-phase microextraction method development and its application in the determination of volatiles in honeys by gas chromatography, Food Chemistry, vol. 126, no. 3, pp , [34] L. Piasenzotto, L. Gracco, and L. Conte, Solid phase microextraction (SPME) applied to honey quality control, Journalofthe Science of Food and Agriculture, vol.83,no.10,pp , [35] E. Alissandrakis, P. A. Tarantilis, P. C. Harizanis, and M. Polissiou, Evaluation of four isolation techniques for honey aroma compounds, the Science of Food and Agriculture, vol. 85,no.1,pp.91 97,2005. [36] E. de la Fuente, I. Martínez-Castro, and J. Sanz, Characterization of Spanish unifloral honeys by solid phase microextraction and gas chromatography-mass spectrometry, Separation Science, vol. 28, no. 9-10, pp , [37] Q. Zhou, C. L. Wintersteen, and K. R. Cadwallader, Identification and quantification of aroma-active components that contribute to the distinct malty flavor of buckwheat honey, Agricultural and Food Chemistry, vol.50,no.7,pp , [38] B. Fickert and P. Schieberle, Identification of the key odorants in barley malt (caramalt) using GC/MS techniques and odour dilution analyses, Die Nahrung,vol.42,no.6,pp ,1998. [39] M. Shimoda, Y. Wu, and Y. Osajima, Aroma compounds from aqueous solution of haze (Rhus succedanea) honey determined by adsorptive column chromatography, Agricultural and Food Chemistry,vol.44,no.12,pp ,1996. [40] R. F. A. Moreira, L. C. Trugo, M. Pietroluongo, and C. A. B. de Maria, Flavor composition of cashew (Anacardium occidentale) andmarmeleiro(croton species) honeys, Agricultural and Food Chemistry, vol. 50, no. 26, pp , [41] C. A. B. de Maria and R. F. A. Moreira, Volatile compounds in floral honeys, Quimica Nova,vol.26,no.1,pp.90 96,2003.

12 Medicinal Chemistry Photoenergy Organic Chemistry International Analytical Chemistry Advances in Physical Chemistry Carbohydrate Chemistry Quantum Chemistry Submit your manuscripts at The Scientific World Journal Inorganic Chemistry Theoretical Chemistry Spectroscopy Analytical Methods in Chemistry Chromatography Research International Electrochemistry Catalysts Applied Chemistry Bioinorganic Chemistry and Applications Chemistry Spectroscopy